The present invention is a continuation-in-part of parent patent application Ser. No. 089,568, filed Aug. 26, 1987.
One class of agents which has been shown to inhibit tumor induction and tumor growth in a number of systems are the glucarolactone compounds. Various glucarolactone-based compounds, including calcium glucarate (CGT), microencapsulated D-glucaro-1,4-lactone, potassium hydrogen glucarate and 2,4-di-0-acetyl-D-glucaro-1-lactone, are known to be effective as inhibitors of beta-glucuronidase in cells, blood, urine and in the intestine and liver. By inhibiting beta-glucuronidase less detoxified (that is glucuronidated) toxins are hydrolysed and therefore more toxins are excreted. As a result, such glucarolactone-based compounds are useful in the treatment and prevention of various types of cancer.
Dietary calcium glucarate, as a slow release form of glucarate, effectively inhibits from 50-70% the chemical induction of tumors in rodent skin, mammary glands, Walaszek et al., Carcinogenesis, 7:1463-1466 (1986), lung, Walaszek et al., Cancer Letters 33:25-32, (1986), and liver, Oredipe et al., Cancer Letters 38:95-99 (1988). (Also, Walaszek, Z. et al. Inhibition of 7,12-dimethylbenzanthracene-induced rat mammary tumorigenesis by 2,5-di-0-acetyl-D-glucaro-1,4:6,3-dilactone, an in-vivo beta-glucuronidase inhibitor. Carcinogenesis 5:767-772, (1984); and, Walaszek, Z., et al., Inhibition of N-methyl-N-nitrosourea-induced mammary tumorigenesis in the rat by a betaglucuronidase inhibitor. IRCS Medical Science 14: 677-678, (1986)).
Glucarate was the active moiety since under the conditions employed, since equimolar calcium as calcium gluconate had no effect. Maximal effect was observed when glucarate was fed throughout the initiation and promotion phases at a level of at least 4% (128 mmol/kg diet). The mechanism of action of glucarate is thought to be enhanced glucuronidation of initiating and promoting agents through equilibrium formation of the beta-glucuronidase inhibitor D-glucaro-1,4-lactone, Levvy, Biochem. J. 52:464-472 (1952).
A second class of agents which has been shown to act at high dosages to inhibit tumor induction and tumor growth in a number of systems are the retinoids, Moon et al., in The Retinoids (M. B. Sporn, A. B. Roberts and D. S. Goodman, Eds.) Vol. 2, pp 327-371, Academic Press, 1984. Retinoid-based compounds including retinylacetate, retinylmethyl ether, 13-cis-retinoic acid and N-(4-hydroxyphenyl) retinaminde (HPR), have similarly been investigated for their anticarcinogenic activity. The inhibition of mammary tumor induction in rats requires near toxic levels of vitamin A, or its metabolites, Hixson et al., Toxicol. Appl. Pharmacol. 47:359-365 (1979). (See also, Abou-Issa, H., et al., Anti-carcinogenic effect of retinoids on 7,12-dimethylbenz(a) anthracene-induced mammary tumor formation and its relation to cyclic AMP-dependent kinase. Biochem. Biophys. Res. Commun. 135: 116-123, (1986); Welsch, C. W., et al., Retinoids and Mammary gland tumorigenesis in Diet, Nutrition and Cancer (B. S. Reddy and L. A. Cohen eds.) CRS Press Boca Raton, FL. pp 1-21, (1986); Schamberger, R. J., Chemoprevention of cancer in Diet, Nutrition and Cancer. (B. S. Reddy and L. A. Cohen eds.) CRC Press, pp. 43-62, (1986); and, Moon, R. C., Inhibition of 7,12-dimethylbenzanthracene-induced mammary carcinogenesis by retinyl acetate. Cancer Res. 36:2626, (1976)).
These studies confirm the activity of relatively high doses of retinoids against the chemical induction of mammary carcinogenesis in the rat. Similarly, high dosages were tested against the chemical carcinogen-mediated induction of tumors in the mammary gland, lung, skin, intestine and liver. Further, retinoids have been shown to protect skin, nasopharnyx, lower respiratory tract, urinary bladder and colon against carcinogens. In addition, these retinoic acid analogs (Vitamin A active compounds) have been tested in combination with the micronutrient selenium.
One problem associated with the use of retinoid-based compounds is that relatively high doses of the retinoids must be administered in order to achieve the desired anticarcinogen effect. Such high doses of retinoids often results in cummulative toxicity, with the excess retinoids being deposited in the liver.
However, there has been no suggestion in the art that a combination of glucarolactone-based compounds and retinoid-based compounds would be especially useful as anticarcinogens; that is, that the combination of these compounds would represent an alternative for use in the prevention of cancer or for use in the therapeutic treatment of cancer.
It has now been found that the use of a combination of glucarolactone-based compounds and retinoid-based compounds or their pharmaceutically-acceptable salts and esters, compounds which are known to be safely administered to humans and animals, significantly reduce the incidence of tumors and further prevent the formation of cancer.
Since glucarate, as end product of glucuronic acid metabolism and a component of fruits and vegetables is normally present in the body at low levels, Matsui et al., Chem. Pharm. Bull (Tokyo) 20:845-848 (1972) and excreted in the urine, we investigated to determine whether it had chemopreventive and/or therapeutic activity in combination with natural metabolites of vitamin A. A synergistic effect between CGT and the synthetic analog N-(4-hydroxyphenyl)retinamide (HPR) which exhibits activity of vitamin A and its metabolites have been observed for both chemoprevention of DMBA-induced rat mammary tumors, Swanson et al., J. Pharmacol. Exptl. Therap. 219:632-637 (1981). The present invention, in part, shows the synergistic effect between CGT and HPR for growth arrest and shrinkage of pre-established rat mammary tumors. The dietary levels at which the synergistic effect is observed were 32-64 mmol/kg diet of CGT and 0.75 mmol/kg diet of HPR. This compares with 128 mmol/kg CGT and 2.00 mmol/kg HRP when used individually. The present invention also shows that the combinations of low non-toxic doses of two natural compounds act synergistically and are effective anti-cancer and chemopreventative agents, both in vivo and in vitro.
It is therefore an object of the present invention to provide a safe and effective method for inhibiting the formation of tumors, inhibiting (or shrinking) tumor growth, and reducing the incidence of cancer in certain high risk populations.
Other objects and advantages of the invention will be apparent from the following detailed description of the invention.